RESUMEN
Long Evans rats were treated by a low-Zinc-Diet (ZD) and the ultrastructure and elemental composition of melanosomes of the RPE and choroidal melanocytes and RPE lipofuscin granules were investigated using Analytical Electron Microscopy (AEM). In controls the Zn mole fraction of melanosomes was 0.05 at% (RPE) and 0.06 at% (choroid), respectively. For ZD-rats the Zn mole fraction of these granules was almost unchanged in the choroid but decreased and was below the detection limit (0.02 at%) in the RPE. ZD-rats produced also giant melanosomes (diameter 1-3 µm) in the choroid and greatly increased amounts of RPE lipofuscin. The giant melanosomes contained about six times as much Cu as control melanosomes. Lipofuscin granules were identified by AEM and could be clearly distinguished from melanosomes by their chemical composition. Thus, changes of the ultrastructure and transition metal storage of melanosomes due to a ZD can be directly traced using AEM.
Asunto(s)
Dieta , Melanocitos/química , Melanosomas/química , Epitelio Pigmentado de la Retina/química , Zinc/análisis , Animales , Coroides/química , Coroides/citología , Cobre/análisis , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Microanálisis por Sonda Electrónica , Lipofuscina/metabolismo , Melanocitos/ultraestructura , Melanosomas/ultraestructura , Microscopía Electrónica de Transmisión , Ratas , Ratas Long-Evans , Epitelio Pigmentado de la Retina/ultraestructura , Espectroscopía de Pérdida de Energía de ElectronesRESUMEN
BACKGROUND: Age-related macular degeneration (AMD) is associated with lipofuscin accumulation whereas the content of melanosomes decreases. Melanosomes are the main storage of zinc in the pigmented tissues. Since the elderly population, as the most affected group for AMD, is prone to zinc deficit, we investigated the chemical and ultrastructural effects of zinc deficiency in pigmented rat eyes after a six-month zinc penury diet. METHODOLOGY/PRINCIPAL FINDINGS: Adult Long Evans (LE) rats were investigated. The control animals were fed with a normal alimentation whereas the zinc-deficiency rats (ZD-LE) were fed with a zinc deficient diet for six months. Quantitative Energy Dispersive X-ray (EDX) microanalysis yielded the zinc mole fractions of melanosomes in the retinal pigment epithelium (RPE). The lateral resolution of the analysis was 100 nm. The zinc mole fractions of melanosomes were significantly smaller in the RPE of ZD-LE rats as compared to the LE control rats. Light, fluorescence and electron microscopy, as well as immunohistochemistry were performed. The numbers of lipofuscin granules in the RPE and of infiltrated cells (Ø>3 µm) found in the choroid were quantified. The number of lipofuscin granules significantly increased in ZD-LE as compared to control rats. Infiltrated cells bigger than 3 µm were only detected in the choroid of ZD-LE animals. Moreover, the thickness of the Bruch's membrane of ZD-LE rats varied between 0.4-3 µm and thin, rangy ED1 positive macrophages were found attached at these sites of Bruch's membrane or even inside it. CONCLUSIONS/SIGNIFICANCE: In pigmented rats, zinc deficiency yielded an accumulation of lipofuscin in the RPE and of large pigmented macrophages in the choroids as well as the appearance of thin, rangy macrophages at Bruch's membrane. Moreover, we showed that a zinc diet reduced the zinc mole fraction of melanosomes in the RPE and modulated the thickness of the Bruch's membrane.
Asunto(s)
Enfermedades Carenciales/metabolismo , Lipofuscina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Zinc/deficiencia , Animales , Microanálisis por Sonda Electrónica , Inmunohistoquímica , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Ratas , Ratas Long-EvansRESUMEN
PURPOSE: Tumor necrosis factor alpha (TNF)-alpha contributes to inflammation-associated angiogenesis. This study investigates the role of TNF-alpha receptors 1a and 1b in the development of choroidal neovascularization (CNV). METHODS: CNV was induced in Tnfrsf1a(-/-) and Tnfrsf1b(-/-) mice with C57Bl6/J background and their wild-type (WT) (C57Bl/6J) controls by laser damage to the Bruch's membrane. TNF-alpha expression in RPE/choroid was determined by Western blot analysis. Pathologic angiogenesis was estimated qualitatively and quantitatively by fluorescein angiography and histology on choroidal flat mounts and paraffin cross-sections. Inflammatory cell invasion was investigated by clodronic acid depletion of circulating macrophages and immunochemistry, and the apoptotic activity was investigated by TUNEL assay and by caspase-3 and caspase-8 expression. Receptor 1b-specific Bmx/Etk kinase was detected by immunochemistry. RESULTS: TNF-alpha levels were elevated after laser treatment. Severe CNV lesions and increased macrophage invasion were observed in Tnfrsf1a(-/-) compared with WT and Tnfrsf1b(-/-) mice. Increased immunoreactivity for Bmx/Etk kinase corresponded to the severity of CNV formation. Reduced pathologic angiogenesis and macrophage invasion in Tnfrsf1b(-/-) mice (vs. WT and Tnfrsf1a(-/-)) was accompanied by enhanced endothelial cell apoptosis and by caspase-3 and caspase-8 activation. CONCLUSIONS: Receptor 1b promotes the recruitment of inflammatory cells to the site of injury and exacerbated pathologic angiogenesis probably by way of the Bmx/Etk-kinase-dependent pathway in the absence of receptor 1a. On the other hand, receptor 1a-dependent apoptosis in the absence of receptor 1b leads to reduced inflammatory response and CNV lesions after laser treatment. This demonstrates the potential for specific targeting of TNF-alpha receptors for future therapies of inflammation-associated choroidal neovascularization.
Asunto(s)
Coroides/metabolismo , Neovascularización Coroidal/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral/fisiología , Epitelio Pigmentado de la Retina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis , Western Blotting , Lámina Basal de la Coroides/cirugía , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Movimiento Celular , Neovascularización Coroidal/diagnóstico , Neovascularización Coroidal/etiología , Angiografía con Fluoresceína , Técnica del Anticuerpo Fluorescente Indirecta , Etiquetado Corte-Fin in Situ , Coagulación con Láser , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Proteínas Tirosina Quinasas/metabolismoRESUMEN
To investigate the effects of zinc supplementation on human amelanotic (ARPE-19) and native pigmented retinal pigment epithelial cells (hRPE) under normal light conditions and after ultraviolet A light exposure. hRPE cells, containing both melanin and lipofuscin granules, were prepared from human donor eyes of 60-70 year old patients. Cells of the amelanotic ARPE-19 cell line and pigmented hRPE cells were treated with zinc chloride and subjected to oxidative stress by UV-A irradiation. Intracellular H(2)O(2) formation was measured using a fluorescence oxidation assay. Additionally, apoptosis and viability assays were performed. Control cells were treated identically except for irradiation and zinc supplementation. Under normal light conditions, zinc treated hRPE cells produced less H(2)O(2) than unsupplemented hRPE cells. Viability and apoptosis events did not change. After UV-A irradiation, ARPE and hRPE cells were greatly impaired in all tests performed compared to the non-irradiated controls. No differences were found after zinc supplementation. hRPE cells showed a higher apoptosis and mortality rate than non-pigmented cells when stressed by UV-A light. ARPE cells never showed any zinc related effects. In contrast, without irradiation, zinc supplementation reduced H(2)O(2) production in pigmented hRPE cells slightly. We did not find any zinc effect in irradiated hRPE cells. After UV light exposure, pigmented cells showed a higher apoptosis and mortality than cells lacking any pigmentation. We conclude that cells with pigmentation consisting of melanin and lipofuscin granules have more prooxidative than antioxidative capacity when stressed by UV light exposure compared to cells lacking any pigmentation.
Asunto(s)
Senescencia Celular , Estrés Oxidativo/efectos de la radiación , Epitelio Pigmentado Ocular/patología , Pigmentación , Rayos Ultravioleta/efectos adversos , Zinc/farmacología , Anciano , Apoptosis , Supervivencia Celular , Humanos , Peróxido de Hidrógeno , Persona de Mediana Edad , Epitelio Pigmentado Ocular/efectos de los fármacos , Epitelio Pigmentado Ocular/efectos de la radiaciónRESUMEN
5,6-dihydroxyindole (DHI) is a melanin pigment precursor with antioxidant properties. In the light of a report about cytotoxicity of DHI, the aim of this study was to assess possible toxic effects of DHI on cells related to the eye, such as human ARPE-19 cells and mouse retinal explants. Moreover, DHI was tested on its effects on retinal function in vivo using electroretinography. We found cytotoxicity of DHI against ARPE-19 cells at 100 microM, but not at 10 microM. 10 microM DHI exhibited a slight, though not significant protective activity against UV-A damage in ARPE-19 cells. We found cytoprotection in cultured mouse retinas by 50 microM DHI or its diacetylated derivative 5,6-diacetoxyindole (DAI), respectively. In ERG measurements in vivo, amplitudes were decreased only slightly by 100 microM DHI compared to saline, whereas a better preservation of amplitudes was visible at 10 microM DHI, in particular with respect to cones. In histological sections, more cones were found at 10 microM DHI than at 100 microM DHI. As a conclusion, DHI shows a slight protective effect at 10 microM both in vitro and in vivo. At 100 microM, it shows a strong cytotoxicity in vitro, which is strongly reduced in vivo.
Asunto(s)
Citoprotección , Indoles/toxicidad , Retina/efectos de los fármacos , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Electrorretinografía , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Retina/patología , Retina/fisiologíaRESUMEN
Low ocular pigmentation and high long-term exposure to bright light are believed to increase the risk of developing age-related macular degeneration (ARMD). To investigate the role of pigmentation during bright light exposure, cell damage in retinae and choroids of pigmented and non-pigmented rats were compared. Pigmented Long Evans (LE) rats and non-pigmented (albino) Wistar rats were exposed to high intensity visible light from a cold light source with 140,000 lux for 30 min. Control animals of both strains were not irradiated. The animals had their pupils dilated to prevent light absorbance by iris pigmentation. 22 h after irradiation, the rats were sacrificed and their eyes enucleated. Posterior segments, containing retina and choroid, were prepared for light and electron microscopy. Twenty different sections of specified and equal areas were examined in every eye. In albino rats severe retinal damage was observed after light exposure, rod outer segments (ROS) were shortened and the thickness of the outer nuclear layer (ONL) was significantly diminished. Choriocapillaris blood vessels were obstructed. In wide areas the retinal pigment epithelium (RPE) was absent in albino rats after irradiation. In contrast, LE rats presented much less cell damage in the RPE and retina after bright light exposure, although intra-individual differences were observed. The thickness of the ONL was almost unchanged compared to controls. ROS were shortened in LE rats, but the effect was considerably less than that seen in the albinos. Only minimal changes were found in choroidal blood vessels of pigmented rats. The RPE showed certain toxic damage, but cells were not destroyed as in the non-pigmented animals. The number of melanin granules in the RPE of LE rats was reduced after irradiation. Ocular melanin protects the retina and choroid of pigmented eyes against light-induced cell toxicity. Physical protection of iris melanin, as possible in eyes with non-dilated pupils, does not seem to play a major role in our setup. Biochemical mechanisms, like reducing oxidative intracellular stress, are more likely to be responsible for melanin-related light protection in eyes with dilated lens aperture.
Asunto(s)
Coroides/irrigación sanguínea , Luz/efectos adversos , Melaninas/farmacología , Vasos Retinianos/efectos de los fármacos , Vasos Retinianos/efectos de la radiación , Animales , Microscopía Electrónica , Ratas , Ratas Long-Evans , Ratas Wistar , Especies Reactivas de Oxígeno/efectos de la radiación , Vasos Retinianos/citología , Vasos Retinianos/ultraestructuraRESUMEN
Tyrosinase (EC 1.14.18.1) is the rate limiting enzyme of melanogenesis and it is unclear whether it is synthesized in postnatal retinal pigment epithelium (RPE). Cultured RPE cells from cattle were fed with isolated rod outer segments (ROS). After phagocytosis, RPE cells were tested for tyrosinase presence and activity with three independent methods: (1) ultrastructural DOPA (l-3,4-dihydroxyphenylalanine) histochemistry (2) immunocytochemistry with anti-tyrosinase antibodies (3) measuring tyrosine hydroxylase activity using [(3)H]tyrosine. With all three methods tyrosinase was found in RPE cells after ROS-feeding but was absent without feeding. In contrast to the classical hypothesis, we demonstrated with three independent methods that the expression of tyrosinase and its enzymatic activity are induced in cultured adult RPE by phagocytosis of rod outer segments (ROS) in vitro.
Asunto(s)
Monofenol Monooxigenasa/biosíntesis , Epitelio Pigmentado Ocular/enzimología , Animales , Anticuerpos/inmunología , Bovinos , Células Cultivadas , Dihidroxifenilalanina/metabolismo , Endosomas/enzimología , Endosomas/ultraestructura , Células Epiteliales/enzimología , Células Epiteliales/ultraestructura , Inmunohistoquímica/métodos , Melanosomas/enzimología , Melanosomas/ultraestructura , Microscopía Electrónica/métodos , Monofenol Monooxigenasa/inmunología , Estrés Oxidativo/fisiología , Fagocitosis/fisiología , Epitelio Pigmentado Ocular/ultraestructura , Segmento Externo de la Célula en Bastón/fisiología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
BACKGROUND: Age-related macular degeneration (AMD) is associated with lower melanin pigmentation and is more prevalent among the elderly Caucasian population than among Africans. A correlation between light iris colour, fundus pigmentation and the incidence of AMD is reported. Moreover, melanin represents the main storage of zinc in the eye. Zinc enhances antioxidant capacity through its function as a cofactor of important enzymes or by influencing gene expression of regulatory elements in the eye. In this study, we investigated the uptake and storage of zinc in the human choroid/retinal pigment epithelium (RPE) complexes in dependence on the fundus pigmentation as judged by the iris colour. MATERIAL AND METHODS: Choroid/RPE complexes of blue and brown human eyes were used. Tissues without any substitution served as controls. Specimens from choroid/RPE complexes were incubated with 100 microM zinc chloride for 24 h. After incubation, pieces of the complexes were stored to investigate the uptake of zinc. The rest of the tissues were kept for 3 and 7 days in culture medium (DMEM) for storage examination. The concentration of zinc was measured by inductively coupled plasma mass spectrometry. RESULTS: After 24 h of zinc treatment the concentration of zinc in the choroid/RPE complexes of blue eyes was not significantly increased. The concentration of zinc in highly pigmented tissues (brown eyes) was increased by the factor 5.1 after 24 h and remained at high levels after 3 days (factor 4.4) and 7 days (factor 2.8). CONCLUSIONS: Zinc uptake in the choroid/RPE complex correlates to the iris colour. Alterations of the degree of iris pigmentation result in differences of zinc uptake and storage in the choroids. A potential protective role of zinc may be more prominent in dark- than in light-coloured eyes.
Asunto(s)
Color del Ojo/fisiología , Epitelio Pigmentado Ocular/metabolismo , Zinc/farmacocinética , Adulto , Anciano , Anciano de 80 o más Años , Coroides/citología , Coroides/efectos de los fármacos , Coroides/metabolismo , Medios de Cultivo , Color del Ojo/efectos de los fármacos , Fondo de Ojo , Humanos , Espectrometría de Masas , Persona de Mediana Edad , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/efectos de los fármacos , Técnicas de Cultivo de TejidosRESUMEN
Age-related macular degeneration (AMD) is more prevalent among the elderly Caucasians than in Africans. A significant association between light iris colour, fundus pigmentation and incidence of AMD is reported, suggesting a possible correlation with melanin pigment. Zinc is known to bind to melanin in pigmented tissues and to enhance antioxidant capacity by function as a cofactor or gene expression factor of antioxidant enzymes in the eye. In this in vitro study, we investigated the uptake and storage of zinc in human irides. Irides of blue and brown human eyes were used. The number of melanocytes was measured. Tissues without any treatment served as controls. The irides were incubated with 100 microM zinc chloride in culture medium for 24 h. Specimens of the tissues were stored for the uptake examination. The remained pieces were further incubated for 3 and 7 d to investigate the storage of zinc. The concentration of zinc was measured by inductively coupled plasma mass spectrometry (ICP-MS). Melanocytes count was significantly higher in the brown tissues (P < 0.0001). Zinc concentration of blue coloured irides after 24 h zinc treatment was close to the controls. We did not observe any significant storing. In contrast, the concentration of zinc in brown irides was significantly increased after 24 h (P < or = 0.01) and remained at a high level for 7 d. The uptake of zinc is likely dependent on the amount of pigmentation in human iris. Therefore, we assume that in patients suffering from AMD the degree of pigmentation of the irides and eventually fundi should be under consideration when the patients are treated with zinc supplementation.